Non-denaturing Fluorescence In Situ Hybridization Research Articles

Karyotyping Dasypyrum breviaristatum chromosomes with multiple oligonucleotide probes reveals the genomic divergence in Dasypyrum.

The perennial species Dasypyrum breviaristatum (genome Vb) contains many potentially valuable genes for the improvement of common wheat. Construction of a detailed karyotype of D. breviaristatum chromosomes will be useful for the detection of Dasypyrum chromatin in wheat background. We established the standard karyotype of 1Vb-7Vb chromosomes through nondenaturing fluorescence in situ hybridization (ND-FISH) technique using 28 oligonucleotide probes from the wheat - D. breviaristatum partial amphiploid TDH-2 (AABBVbVb) and newly identified wheat - D. breviaristatum disomic translocation and addition lines D2138 (6VbS.2VbL), D2547 (4Vb), and D2532 (3VbS.6VbL) by comparative molecular marker analysis. The ND-FISH with multiple oligo probes was conducted on the durum wheat - D. villosum amphiploid TDV-1 and large karyotype differences between D. breviaristatum and D. villosum was revealed. These ND-FISH probes will be valuable for screening the wheat - Dasypyrum derivative lines for chromosome identification, and the newly developed wheat - D. breviaristatum addition lines may broaden the gene pool of wheat breeding. The differences between D. villosum and D. breviaristatum chromosomes revealed by ND-FISH will help us understand evolutionary divergence of repetitive sequences within the genus Dasypyrum.

New evidence confirming the CD genomic constitutions of the tetraploid Avena species in the section Pachycarpa Baum.

The tetraploid Avena species in the section Pachycarpa Baum, including A. insularis, A. maroccana, and A. murphyi, are thought to be involved in the evolution of hexaploid oats; however, their genome designations are still being debated. Repetitive DNA sequences play an important role in genome structuring and evolution, so understanding the chromosomal organization and distribution of these sequences in Avena species could provide valuable information concerning genome evolution in this genus. In this study, the chromosomal organizations and distributions of six repetitive DNA sequences (including three SSR motifs (TTC, AAC, CAG), one 5S rRNA gene fragment, and two oat A and C genome specific repeats) were investigated using non-denaturing fluorescence in situ hybridization (ND-FISH) in the three tetraploid species mentioned above and in two hexaploid oat species. Preferential distribution of the SSRs in centromeric regions was seen in the A and D genomes, whereas few signals were detected in the C genomes. Some intergenomic translocations were observed in the tetraploids; such translocations were also detected between the C and D genomes in the hexaploids. These results provide robust evidence for the presence of the D genome in all three tetraploids, strongly suggesting that the genomic constitution of these species is DC and not AC, as had been thought previously.

An efficient Oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae.

A chromosome-specific painting technique has been developed which combines the most recent approaches of the companion disciplines of molecular cytogenetics and genome research. We developed seven oligonucleotide (oligo) pools derivd from single-copy sequences on chromosomes 1 to 7 of barley (Hordeum vulgare L.) and corresponding collinear regions of wheat (Triticum aestivum L.). The seven groups of pooled oligos comprised between 10986 and 12496 45-bp monomers, and these then produced stable fluorescence in situ hybridization (FISH) signals on chromosomes of each linkage group of wheat and barley. The pooled oligo probes were applied to high-throughput karyotyping of the chromosomes of other Triticeae species in the genera Secale, Aegilops, Thinopyrum, and Dasypyrum, and the study also extended to some wheat-alien amphiploids and derived lines. We demonstrated that a complete set of whole-chromosome oligo painting probes facilitated the study of inter-species chromosome homologous relationships and visualized non-homologous chromosomal rearrangements in Triticeae species and some wheat-alien species derivatives. When combined with other non-denaturing FISH procedures using tandem-repeat oligos, the newly developed oligo painting techniques provide an efficient tool for the study of chromosome structure, organization, and evolution among any wild Triticeae species with non-sequenced genomes.

Generation and molecular marker and cytological characterization of wheat - Secale strictum subsp. anatolicum derivatives.

Cereal rye and its wild forms are important sources of genetic diversity for wheat breeding due to their resistances to biotic and abiotic stresses. Secale strictum subsp. anatolicum (Boiss.) K. Hammer (SSA) is a weedy relative of cultivated rye, S. cereale. Meiotic chromosome pairing in F1 hybrids of SSA and S. cereale reveals strong genomic affinity between the two genomes. A study of the transferability of S. cereale sequence-based markers to SSA and hexaploid triticale demonstrated their applicability for tracing SSA chromatin in wheat. The transferability of the markers was over 80% from homoeologous groups 1, 2, and 3, and greater than 70% from groups 4 to 7. This study focused on the generation and molecular and cytogenetic characterization of wheat-SSA alien derivatives. Twelve were identified using combinations of non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and molecular marker analysis. All SSA chromosomes, except 3Ra and 6Ra, were transferred to wheat either in the form of monosomic additions (MA), mono-telosomic additions (MtA), double-mono-telosomic additions (dMtA), or double-monosomic additions (dMA). The germplasm developed in this study will help to enhance the genetic base of wheat and facilitate molecular breeding of wheat and triticale.

Molecular dissection of Secale africanum chromosome 6Rafr in wheat enabled localization of genes for resistance to powdery mildew and stripe rust

BackgroundIntrogression of chromatin from Secale species into common wheat has for decades been a successful strategy for controlling the wheat diseases. The wild Secale species, Secale africanum Stapf., is a valuable source for resistance to foliar disease of wheat. A wheat-S. africanum chromosome 6Rafr substitution line displayed resistance to both powdery mildew and stripe rust at the adult-plant stage.ResultsWheat-S. africanum chromosome 6Rafr deletion and translocation lines were produced and identified by sequential non-denaturing fluorescence in situ hybridization (ND-FISH) using multiple Oligo-based probes. Different ND-FISH patterns were observed between S. cereale 6R and S. africanum 6Rafr. With reference to the physical map of the draft genome sequence of rye inbred line Lo7, a comprehensive PCR marker analysis indicated that insertions and deletions had occurred by random exchange between chromosomes 6R and 6Rafr. A survey of the wheat- S. africanum 6Rafr lines for disease resistance indicated that a powdery mildew resistance gene(s) was present on the long arm of 6Rafr at FL0.85–1.00, and that a stripe rust resistance gene(s) was located in the terminal region of 6RafrS at FL0.95–1.00. The wheat-S. africanum 6Rafr introgression lines also displayed superior agronomic traits, indicating that the chromosome 6Rafr may have little linkage drag in the wheat background.ConclusionsThe combination of molecular and cytogenetic methods allowed to precisely identify the chromosome rearrangements in wheat- S. africanum 6Rafr substitution, deletion and translocation lines, and compare the structural difference between chromosomes 6R and 6Rafr. The wheat- S. africanum 6Rafr lines containing gene(s) for powdery mildew and stripe rust resistance could be used as novel germplasm for wheat breeding by chromosome engineering.

Development and molecular cytogenetic characterization of Thinopyrum bessarabicum introgression lines in hexaploid and tetraploid wheats.

A variety of Thinopyrum bessarabicum introgressions in both hexaploid and tetraploid wheats were generated and characterized by molecular cytogenetic analysis. Six wheat-J genome recombinants were identified with ND-FISH and GISH. Diploid wheatgrass, Thinopyrum bessarabicum (2n = 2x = 14, EbEb or JbJb or JJ), is a well-known alien source of salinity tolerance and disease resistance for wheat improvement. The true genetic potential and effect of such introgressions into wheat can be best studied in chromosomal addition or substitution lines. Here, we report the generation and characterization of various categories of Th. bessarabicum derivatives in both hexaploid and tetraploid cultivated wheats. Sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and genomic in situ hybridization (GISH) are robust techniques to visualize the size of alien introgressions and breakpoints. We identified a complete set of monosomic addition lines into both bread wheat and durum wheat, except for 7J in durum wheat, by sequential ND-FISH and GISH. We also characterized alien derivatives belonging to various classes including mono-telosomic additions, disomic additions, monosomic substitutions, double monosomic substitutions, monosomic substitution-monosomic additions, double monosomic additions, and multiple monosomic additions into both bread and durum wheats. In addition, various wheat-Th. bessarabicum recombinant chromosomes were also detected in six alien derivatives. These wheat-Th. bessarabicum derivatives will provide useful cytogenetic resources for improvement of both hexaploid and tetraploid wheats.

ND-FISH-positive oligonucleotide probes for detecting specific segments of rye (Secale cereale L.) chromosomes and new tandem repeats in rye

Rye (Secale cereale L.) has been widely used to improve wheat (Triticum aestivum L.) cultivars. Oligo probes combined with non-denaturing fluorescence in situ hybridization (ND-FISH) technology provide a convenient and efficient way to identify individual rye chromosomes. However, suitable ND-FISH-positive oligo probes for recognizing specific segments of rye chromosomes are lacking. Five new ND-FISH-positive oligo probes: Oligo-5BL.46, Oligo-5A8080, Oligo-5A8080.1, Oligo-1AL.73, and Oligo-0R3, combined with two previously reported oligo probes, Oligo-44 and Oligo-45, were used in this study. Probes Oligo-5BL.46, Oligo-5A8080 and Oligo-44 produced signals only in intercalary regions of arms 1RS, 5RS, and 5RL, respectively. Probe Oligo-5A8080.1 combined with probe Oligo-45 distinguished the intercalary regions of arms 1RS, 5RS, and 6RS simultaneously. Oligo-5A8080 and Oligo-5A8080.1 revealed variation in the distribution of 5S rDNA sequences and polymorphism among 5R chromosomes. Probe Oligo-1AL.73 produced signals only on chromosomes 4R and 7R and contributed to the construction of an improved FISH map of chromosome 4RKu and to the confirmation of 4RLKu breakpoints in wheat-rye 4RLKu translocation chromosomes. Oligo-0R3 produced signals in the telomeric and subtelomeric regions of 14 rye chromosomes. These oligo probes also revealed five new tandem repeats in rye. Using the oligo probes reported in this study, the short arms of 1R, 5R, and 6R and the long arms of 4R and 7R can be easily discriminated when these chromosomes are broken.

The production of wheat – Aegilops sharonensis 1Ssh chromosome substitution lines harboring alien novel high-molecular-weight glutenin subunits

In our previous work, a novel high-molecular-weight glutenin subunit (HMW-GS) with an extremely large molecular weight from Aegilops sharonensis was identified that may contribute to excellent wheat (Triticum aestivum) processing quality and increased dough strength, and we further generated HMW-GS homozygous lines by crossing. In this study, we crossed the HMW-GS homozygous line 66-17-52 with 'Chinese Spring' Ph1 mutant CS ph1b to induce chromosome recombination between wheat and Ae. sharonensis. SDS-PAGE was used to identify 19 derived F2 lines with the HMW-GSs of Ae sharonensis. The results of non-denaturing fluorescence in situ hybridization (ND-FISH) indicated that lines 6-1 and 6-7 possessed a substitution of both 5D chromosomes by a pair of 1Ssh chromosomes. Further verification by newly developed 1Ssh-specific chromosome markers showed that these two lines amplified the expected fragment. Thus, it was concluded that lines 6-1 and 6-7 are 1Ssh(5D) chromosome substitution lines. The 1Ssh(5D) chromosome substitution lines, possessing alien subunits with satisfactory quality-associated structural features of large repetitive domains and increased number of subunits, may have great potential in strengthening the viscosity and elasticity of dough made from wheat flour. Therefore, these substitution lines can be used for wheat quality improvement and further production of 1Ssh translocation lines.

Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor

Su1-Ph1, which we previously introgressed into wheat from Aegilops speltoides, is a potent suppressor of Ph1 and a valuable tool for gene introgression in tetraploid wheat. We previously introgressed Su1-Ph1, a suppressor of the wheat Ph1 gene, from Aegilops speltoides into durum wheat cv Langdon (LDN). Here, we evaluated the utility of the introgressed suppressor for inducing introgression of alien germplasm into durum wheat. We built LDN plants heterozygous for Su1-Ph1 that simultaneously contained a single LDN chromosome 5B and a single Ae. searsii chromosome 5Sse, which targeted them for recombination. We genotyped 28 BC1F1 and 84 F2 progeny with the wheat 90-K Illumina single-nucleotide polymorphism assay and detected extensive recombination between the two chromosomes, which we confirmed by non-denaturing fluorescence in situ hybridization (ND-FISH). We constructed BC1F1 and F2 genetic maps that were 65.31 and 63.71cM long, respectively. Recombination rates between the 5B and 5Sse chromosomes were double the expected rate computed from their meiotic pairing, which we attributed to selection against aneuploid gametes. Recombination rate between 5B and 5Sse was depressed compared to that between 5B chromosomes in the proximal region of the long arm. We integrated ND-FISH signals into the genetic map and constructed a physical map, which we used to map a 172,188,453-bp Ph1 region. Despite the location of the region in a low-recombination region of the 5B chromosome, we detected three crossovers in it. Our data show that Su1-Ph1 is a valuable tool for gene introgression and gene mapping based on recombination between homoeologous chromosomes in wheat.

Molecular cytogenetic characterization of stem rust and stripe rust resistance in wheat-Thinopyrum bessarabicum–derived doubled haploid lines

Thinopyrum species are potential sources of disease resistance for wheat. Thinopyrum bessarabicum is a well-known source of salt tolerance, but there are very few reports of rust resistance being transferred from this species to wheat. Here, we report wheat-Th. bessarabicum–derived stem rust and stripe rust–resistant doubled haploid (DH) lines and characterized using molecular markers and cytogenetic analyses. We used sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and genomic in situ hybridization (GISH) to determine their genomic compositions. Cytological results were validated by molecular analysis using genome-specific PCR markers. These lines were either alien additions, partial diploids, or partial amphidiploids. Line DH9 was a disomic addition line carrying chromosome 4Jb whereas DH1 was a double disomic addition line for chromosomes 6Jb and 7Jb. Lines DH7 and DH8 were partial diploids that were cytologically unstable due to having ph1ph1 genotype. DH7 also had a pair of T6BL-6JbL.6JbS translocated chromosomes and was monosomic for a T5DS-5JbS.5JbL translocation. The size of the translocated alien segment in chromosome T5DS-5JbS.5JbL was L+(0–0.42)S, whereas the size of wheat chromatin in chromosome T6BL-6JbL.6JbS was S+(0–0.85)L. Lines DH5 (2n = 50) and DH11 (2n = 54) were partial amphidiploids with eight and 12 Th. bessarabicum chromosomes, respectively. Lines DH7, DH8, and DH11 expressed an intermediate level of resistance to stem rust with infection types (ITs) varying from 1+2 to 23-, whereas they were immune or near-immune (IT 0 to 0) to stripe rust. Comparison of the rust reactions, genome compositions, and molecular markers of the DH lines along with their parents indicated that the Thinopyrum chromosome conferring resistance to both rusts was 2Jb.

Diversified Chromosome Rearrangements Detected in a Wheat‒Dasypyrum breviaristatum Substitution Line Induced by Gamma-Ray Irradiation.

To determine the composition of chromosome aberrations in a wheat‒Dasypyrum breviaristatum substitution line with seeds treated by a dose of gamma-rays (200 Gy), sequential non-denaturing fluorescence in situ hybridization (ND-FISH) with multiple oligonucleotide probes was used to screen individual plants of the mutagenized progenies. We identified 122 types of chromosome rearrangements, including centromeric, telomeric, and intercalary chromosome translocations from a total of 772 M1 and 872 M2 plants. The frequency of reciprocal translocations between B- and D-chromosomes was higher than that between A- and D-chromosomes. Eight translocations between D. breviaristatum and wheat chromosomes were also detected. The 13 stable plants with multiple chromosome translocations displayed novel agronomic traits. The newly developed materials will enhance wheat breeding programs through wheat‒Dasypyrum introgression and also facilitate future studies on the genetic and epigenetic effects of translocations in wheat genomics.

Characterization of a new hexaploid triticale 6D(6A) substitution line with increased grain weight and decreased spikelet number

Hexaploid triticale (×Triticosecale, AABBRR) is an important forage crop and a promising energy plant. Transferring D-genome chromosomes or segments from common wheat (Triticum aestivum) into hexaploid triticale is attractive in improving its economically important traits. Here, a hexaploid triticale 6D(6A) substitution line Lin 456 derived from the cross between the octoploid triticale line H400 and the hexaploid wheat Lin 56 was identified and analyzed by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and molecular markers. The GISH analysis showed that Lin 456 is a hexaploid triticale with 14 rye (Secale cereale) chromosomes and 28 wheat chromosomes, whereas non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular marker analysis revealed that it is a 6D(6A) substitution line. In contrast to previous studies, the signal of Oligo-pSc119.2 was observed at the distal end of 6DL in Lin 456. The wheat chromosome 6D was associated with increased grain weight and decreased spikelet number using the genotypic data combined with the phenotypes of the F2 population in the three environments. The thousand-grain weight and grain width in the substitution individuals were significantly higher than those in the non-substitution individuals in the F2 population across the three environments. We propose that the hexaploid triticale 6D(6A) substitution line Lin 456 can be a valuable and promising donor stock for genetic improvement during triticale breeding.

New ND-FISH-Positive Oligo Probes for Identifying Thinopyrum Chromosomes in Wheat Backgrounds.

Thinopyrum has been widely used to improve wheat (Triticum aestivum L.) cultivars. Non-denaturing fluorescence in situ hybridization (ND-FISH) technology using oligonucleotides (oligo) as probes provides a convenient and efficient way to identify alien chromosomes in wheat backgrounds. However, suitable ND-FISH-positive oligo probes for distinguishing Thinopyrum chromosomes from wheat are lacking. Two oligo probes, Oligo-B11 and Oligo-pThp3.93, were designed according to the published Thinopyrum ponticum (Th. ponticum)-specific repetitive sequences. Both Oligo-B11 and Oligo-pThp3.93 can be used for ND-FISH analysis and can replace conventional GISH and FISH to discriminate some chromosomes of Th. elongatum, Th. intermedium, and Th. ponticum in wheat backgrounds. The two oligo probes provide a convenient way for the utilization of Thinopyrum germplasms in future wheat breeding programs.

The Polymorphisms of Oligonucleotide Probes in Wheat Cultivars Determined by ND-FISH.

Non-denaturing fluorescence in situ hybridization (ND-FISH) has been used to distinguish wheat chromosomes and to detect alien chromosomes in the wheat genome. In this study, five different oligonucleotide probes were used with ND-FISH to examine 21 wheat cultivars and lines. These oligonucleotide probes distinguished 42 wheat chromosomes and also detected rye chromatin in the wheat genome. Moreover, the signal patterns of the oligonucleotide probes Oligo-pTa535-1 and Oligo-pSc119.2-1 showed high polymorphism in the wheat chromosomes. A total of 17.6% of the A group chromosomes, 25.9% of the B group chromosomes and 8.9% of the D group chromosomes showed obvious mutations when they were compared to the standard ND-FISH signal patterns, and most of them were Oligo-pSc119.2-1 mutants. The results suggested that these polymorphisms could be induced by the crossing of wheat cultivars. The results provided more information for the further application of oligonucleotide probes and ND-FISH.

Stable expression and heredity of alien Glu-1Ssh in wheat-Aegilops sharonensis hybrid progenies

In our previous study, we have isolated novel HMW-GSs with unusually large molecular weight from Aegilops sharonensis; and to introduce this novel HMW-GSs variant into common wheat, we produced a number of amphiploids between various tetraploid wheat, hexaploid wheat and Ae. sharonensis by distant hybridization. In this study, we reported that the characterization of 24 HMW-GSs homozygous lines from wheat-Ae. sharonensis populations derived by continuous backcross or self-cross to common wheat cultivar LM3, which possessed homozygous HMW-GSs compositions including alien HMW-GS of Ae. sharonensis plus B and/or D genomes encoded HMW-GSs of LM3. According to the profiling of HMW-GS, these 24 homozygous lines can be summarized as four types. The results of Non-denaturing fluorescence in situ hybridization (ND-FISH) indicated that the chromosome numbers of eight of 24 lines were 40, 42, 44 or 45, and the number of alien Ae. sharonensis Ssh were between two and eight. Three 1S(1A) substitution lines, two 1S(1B) substitution lines, three 1S(1D) substitution lines. For the four lines with 44 and 45 chromosomes, they exhibit novel cytogenetic characteristics in which the substitution of 1S(1B) or 1S(1D) and addition of Ae. sharonensis 3S or 4S simultaneously appeared. The 24 homozygous progenies were similar to common wheat LM3 on agronomic traits. Specially, four lines of them showed almost identical agronomic traits to recurrent parent common wheat LM3 than the rest lines. These homozygous progenies could be used not only to evaluate function on quality of single HMW-GS but also to create translocation lines.

Genome-Wide Distribution of Novel Ta-3A1 Mini-Satellite Repeats and Its Use for Chromosome Identification in Wheat and Related Species

A large proportion of the genomes of grasses is comprised of tandem repeats (TRs), which include satellite DNA. A mini-satellite DNA sequence with a length of 44 bp, named Ta-3A1, was found to be highly accumulated in wheat genome, as revealed by a comprehensive sequence analysis. The physical distribution of Ta-3A1 in chromosomes 3A, 5A, 5B, 5D, and 7A of wheat was confirmed by nondenaturing fluorescence in situ hybridization (ND-FISH) after labeling the oligonucleotide probe. The analysis of monomer variants indicated that rapid sequence amplification of Ta-3A1 occurred first on chromosomes of linkage group 5, then groups 3 and 7. Comparative ND-FISH analysis suggested that rapid changes occurred in copy number and chromosomal locations of Ta-3A1 among the different species in the tribe Triticeae, which may have been associated with chromosomal rearrangements during speciation and polyploidization. The labeling and subsequent use of Ta-3A1 by ND-FISH may assist in the precise identification and documentation of novel wheat germplasm engineered by chromosome manipulation.

Characterization of Chromosomal Rearrangement in New Wheat—Thinopyrum intermedium Addition Lines Carrying Thinopyrum—Specific Grain Hardness Genes

The wild species, Thinopyrum intermedium. (Genome StStJSJSJJ), serves as a valuable germplasm resource providing novel genes for wheat improvement. In the current study, non-denaturing fluorescence in situ hybridization (ND-FISH) with multiple probes and comparative molecular markers were applied to characterize two wheat-Th. intermedium chromosome additions. Sequential ND-FISH with new labeled Th. intermedium specific oligo-probes were used to precisely determine the chromosomal constitution of Th. intermedium, wheat—Th. intermedium partial amphiploids and addition lines Hy36 and Hy37. The ND-FISH results showed that the added JS-St translocated chromosomes in Hy36 had minor Oligo-5S ribosomal DNA (rDNA) signals at the short arm, while a pair of J-St chromosomes in Hy37 had major Oligo-pTa71 and minor Oligo-5S rDNA signals. The 90K SNP array and PCR-based molecular markers that mapped on wheat linkage group 5 and 3 facilitated the identification of Thinopyrum chromosome introgressions in the addition lines, and confirmed that added chromosomes in Hy36 and Hy37 were 5JSS.3StS and 5JS.3StS, respectively. Complete coding sequences at the paralogous puroindoline-a (Pina) loci from Th. intermedium were cloned and localized on the short arm of chromosome 5JS of Hy36. Line Hy36 showed a reduction in the hardness index, which suggested that Th. intermedium-specific Pina gene sequences may be associated with the softness trait in wheat background. The molecular cytogenetic identification of novel wheat—Th. intermedium derivatives indicated that the frequent chromosome rearrangement occurred in the progenies of wheat-Thinopyrum hybridization. The new wheat-Thinopyrum derived lines may increase the genetic diversity for wheat breeding.

FISH Karyotype of 85 Common Wheat Cultivars/Lines Displayed by ND-FISH Using Oligonucleotide Probes

Fluorescence in situ hybridization (FISH) can reveal minor structural differences of chromosomes. The karyotype of common wheat (Triticum aestivum L.) based on FISH pattern is seldom reported. In this study, non-denaturing FISH (ND-FISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and (AAG)6 as probes was used to investigate the chromosomal structure of 85 common wheat including 83 wheat-rye 1RS.1BL translocation cultivars/lines, a wheatrye 1RS.1AL translocation cultivar Amigo and Chinese Spring (CS). Two, three, two, three, six, three and four structural types respectively for 1A, 2A, 3A, 4A, 5A, 6A and 7A chromosomes were observed. Two, eight, two, two, four and six types of chromosome for 2B, 3B, 4B, 5B, 6B and 7B were respectively detected. The structure of 1B chromosomes in Amigo and CS is different. Five, two, two and two types of chromosomal structure respectively for 1D, 2D, 3D and 5D were distinguished. Polymorphisms of 1RS.1BL, 4D, 6D and 7D chromosomes were not detected. Chromosomes 1AI, 2AI, 3AI, 4AI, 5AIII, 6AI, 7AIII, 2BI, 3BV, 4BI, 5BII, 6BIII, 7BI, 1DIV, 2DI, 3DI and 5DII appeared in these 85 wheat cultivars/lines at high frequency. Each of the 85 wheat cultivars/lines has a unique karyotype. Amigo is a complex translocation cultivar. The FISH karyotype of wheat chromosomes built in this study provide a reference for the future analyzing wheat genetic stocks and help to learn structural variations of wheat chromosomes. In addition, the results in this study indicate that oligonucleotide probes and ND-FISH technology can be used to identify individual wheat cultivar.

Oligonucleotides and ND-FISH Displaying Different Arrangements of Tandem Repeats and Identification of Dasypyrum villosum Chromosomes in Wheat Backgrounds.

Oligonucleotide probes and the non-denaturing fluorescence in situ hybridization (ND-FISH) technique are widely used to analyze plant chromosomes because they are convenient tools. New oligonucleotide probes, Oligo-Ku, Oligo-3B117.1, Oligo-3B117.2, Oligo-3B117.2.1, Oligo-3B117.3, Oligo-3B117.4, Oligo-3B117.5, Oligo-3B117.6, Oligo-pTa71A-1, Oligo-pTa71A-2, Oligo-pTa71B-1, Oligo-pTa71B-2, Oligo-pTa71C-1, Oligo-pTa71C-2, Oligo-pTa71C-3 and Oligo-pTa71D were designed based on the repetitive sequences KU.D15.15, pSc119.2-like sequence 3B117 and pTa71. Oligonucleotide probe (GT)7 was also used. Oligo-Ku and (GT)7 can be together used to identify Dasypyrum villosum from wheat chromosomes and to distinguish individual D. villosum chromosomes. The oligonucleotide probes that were derived from the same repeat sequence displayed different signal intensity and hybridization sites on the same chromosomes. Both the length and the nucleotide composition of oligonucleotide probes determined their signal intensity. For example, Oligo-3B117.2 (25 bp) and Oligo-pTa71A-2 (46 bp) produced the strongest signals on chromosomes of wheat (Triticum aestivum L.), rye (Secale cereale L.), barley (Hordeum vulgare ssp. vulgare) or D. villosum, the signal of Oligo-3B117.4 (18 bp) on the short arm of 7B chromosome was weaker than that of Oligo-3B117.2.1 (15 bp) and Oligo-3B117.3 (16 bp), and Oligo-pTa71A-1 (38 bp) produced the same strong signals as Oligo-pTa71A-2 did on 1B and 6B chromosomes, but its signals on 1R and 1V chromosomes were weaker than the ones of Oligo-pTa71A-2. Oligonucleotide probes and ND-FISH analysis can reflect the distribution and structural statues of different segments of tandem repeats on chromosomes. The possible reasons why different segments derived from the same repeat sequence produced different signal patterns are discussed.

A simple and efficient non-denaturing FISH method for maize chromosome differentiation using single-strand oligonucleotide probes.

Single-strand oligonucleotides (SSONs hereafter) as probes are becoming a powerful method of chromosome painting in many species. In this study, nine SSONs ((ACT)10, (ACT)19, Knob-1, Knob-2, Knob-3, CentC69-1, MR68-3, K10-72-1, and TR1-357-2) were developed and used for chromosome identification in 16 maize (Zea mays L., 2n = 20) inbred lines and hybrids by non-denaturing fluorescence in situ hybridization (ND-FISH). Each SSON produced clear signals on 2-10 chromosomes of inbred lines B73 and Mo17. A multiplex probe set containing four SSONs ((ACT)10, Knob-2, CentC69-1, and MR68-3) clearly characterized all maize chromosomes in the 16 lines by a single round of ND-FISH and revealed genetic variation at a chromosome level. For example, unique signals on chromosome 6 clearly distinguished all 16 genotypes. The SSONs and multiplex probe developed in this research will facilitate genotype identification and chromosome research in maize.